1. Field of the Invention
The present invention relates to methods for increasing expression of genes encoding polypeptides in a fungal cell.
2. Description of the Related Art
The recombinant production of a native or foreign polypeptide in a fungal host cell. e.g., a yeast or filamentous fungal cell, may provide for a more desirable vehicle for producing the polypeptide in commercially relevant quantities.
Recombinant production of a native or foreign polypeptide is generally accomplished by constructing an expression cassette in which the DNA coding for the polypeptide is placed under the expression control of a promoter from a regulated gene. The expression cassette is introduced into the host cell, usually by plasmid-mediated transformation. Production of the polypeptide is then achieved by culturing the transformed host cell under inducing conditions necessary for the proper functioning of the promoter contained on the expression cassette.
The development of new expression constructs and vectors for the recombinant production of polypeptides in a fungal host cell generally requires the availability of efficient promoters that are suitable for controlling expression of the polypeptides in the host cell. However, even the best known promoters can be inefficient for expressing a gene of interest.
Many promoters are susceptible to being modulated which can increase their efficiency. The Saccharomyces cerevisiae metallothionein gene, designated CUP1, is transcriptionally activated by copper through a specific promoter region, UASCUP1 (upstream activation sequence), reportedly located between −105 and −230 with respect to the CUP1 transcription activation site (Thiele and Hamer, 1986, Mol. Cell. Biol. 6: 1158-1163; Zhou and Thiele, 1993, Biofactors 4: 105-115). The Ace1 protein (Ace1p) of Saccharomyces cerevisiae is responsible for induction of the yeast metallothionein gene CUP1, in the presence of copper ions (Thiele, 1988, Mol. Cell. Biol. 8: 2745-2752) or silver ions (Furst et al., 1988, Cell 55: 705-717). The amino-terminal half of the Ace1p is rich in basic amino acid residues and cysteines and specifically binds to the CUP1 upstream activator sequence in the presence, but not in the absence, of Cu(I) or Ag(I) (Furst et al., 1988, supra). Thiele and Hamer, 1986, Molecular and Cellular Biology 6: 1158-1163, disclose that tandemly duplicated upstream control sequences mediate copper-induced transcription of the Saccharomyces cerevisiae copper-metallothionein gene and a synthetic version of one of these elements confers copper induction on a heterologous promoter when present in two tandem copies.
Gralla et al., 1991, PNAS USA 88: 8558-8562, disclose that Ace1p activates expression of a yeast copper, zinc superoxide dismutase gene. Lapinskas et al., 1993, Current Genetics 24: 388-393, disclose that Ace1p activates expression of a Saccharomyces cerevisiae cytosolic catalase gene.
Mehra et al., 1989, J. Biological Chemistry 264: 19747-19753, describe the cloning and sequences of metallothionein genes from Candida glabrata. Zhou and Thiele, 1991, PNAS USA 88: 6112-6116, describe the isolation of a metal-activated transcription factor gene from Candida glabrata. Thorvaldsen et al., 1993, J. Biological Chemistry 268: 12512-12518, disclose the regulation of the Candida glabrata metallothionein genes, designated MTI, MTIIa, and MTIIb, by AMT1.
Mascorro-Gallardo et al., 1996, Gene 172: 169-170, disclose construction of a CUP1 promoter-based vector to modulate gene expression in Saccharomyces cerevisiae. Macreadie et al., 1989, Plasmid 21: 147-150, disclose a series of yeast expression vectors utilizing the CUP1 gene of Saccharomyces cerevisiae. Hottiger et al., 1994, Yeast 10: 283-296, disclose the physiological characterization of the CUP1 promoter and consequences of overexpressing its transcriptional activator Ace1p.
It is an object of the present invention to provide improved methods for producing a polypeptide in a fungal host cell.